An autonomously replicating sequence from HeLa DNA shows a similar organization to the yeast ARS1 element

Summary A HeLa DNA fragment, which may function as an anchorage point to the nuclear matrix for human chromosomes 1 and 2, also functions as an autonomously replicating sequence (ARS) in the yeast Saccharomyces cerevisiae. In the present report we show that this DNA fragment contains both bent DNA and an A-T rich region which appear to be associated with the ARS function. More interestingly, DNA sequence analysis shows that the spatial distribution of these features is strikingly similar to that found in the yeast ARS1 element.     

Trichoderma reesei sequences that bind to the nuclear matrix enhance transformation frequency

Three DNA fragments, trs1, 2 and 3, were isolated from the Trichoderma reesei genome on the basis of their ability to promote autonomous replication of plasmids in Saccharomyces cerevisiae. Each trs element bound specifically to the isolated T. reesei nuclear matrix in vitro, and two of them bound in vivo, indicating that they are matrix attachment regions (MARs). A similar sequence previously isolated from Aspergillus nidulans (ans1) was also shown to bind specifically to the T. reesei nuclear matrix in vitro. The T. reesei MARs are AT-rich sequences containing 70%, 86% and 73% A+T over 2.9, 0.8 and 3.7 kb, respectively for trs1, 2 and 3. They exhibited no significant sequence homology, but were shown to contain a number of sequence motifs that occur frequently in many MARs identified in other eukaryotes. However, these motifs occurred as frequently in the trs elements as in randomly generated sequences with the same A+T content. trs1 and 3 were shown to be present as single copies in the T. reesei genome. The presence of the trs elements in transforming plasmids enhanced the frequency of integrative transformation of T. reesei up to five fold over plasmids without a trs. No evidence was obtained to suggest that the trs elements promoted efficient replication of plasmids in T. reseei. A mechanism for the enhancement of transformation frequency by the trs elements is proposed. Received: 1 March 1997 / Accepted: 13 May 1997     

In addition to the ARS core, the ARS box is necessary for autonomously replicating sequences

Summary Autonomously replicating sequences (ARSs) were cloned from nuclear and mitochondrial DNA of D. melanogaster using YIp5, which is composed of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^- yeast strain as the recipient. The nucleotide sequences of six ARSs, two from nuclear bulk, two from the nuclear 1.688 satellite, and two from mitochondorial DNA, were determined. The relationship between the transformation frequency and the inclusion of the ARS core, 5 _T ^A TT-TAT _A ^G TTT _T ^A 3, of these fragments was analysed. All the ARSs contained an ARS core or a single base change of it. However, not all the fragments that contained a single base change of the ARS core were able to transform the recipient cells, suggesting that certain bases in the ARS core were not exchangeable. It is suggested by transformation experiments with subfragments that in addition to an ARS core, an ARS box which is located within 25 bp upstream of the ARS core and whose sequence is composed of 5TNT _G ^A AA 3, is necessary for autonomous replication.     

Investigation of mutant frequency at the HPRT locus and changes in microsatellite sequences in healthy young adults

In an attempt to understand the inter-individual variation that occurs in in vivo mutant frequency at the HPRT locus, we have examined the effect of polymorphisms in genes for metabolic enzymes on the mutation rate. In the same population of human volunteers, the background variant frequency in a number of microsatellite sequences was studied to determine individual variation in the capacity to repair mismatches in these sequences. The HPRT mutant frequency of T-cells isolated from a group of 49 healthy, non-smoking adults varied from 0.25 to 9.64×10⁻⁶. The frequency of polymorphisms in CYP1A1, GSTM1 and NAT2 among these individuals was similar to those published, and when subjected to univariate analysis these polymorphisms showed no influence on the HPRT mutant frequency. However, there was a significant interaction between the GSTM1 null genotype and the slow acetylator status in NAT2 (P<0.05) which was associated with higher mutant frequency. Analysis of 30 microsatellite sequences in 20 HPRT proficient clones per individual showed only six alterations in total, giving an overall mutation rate per allele of 0.01%, whilst three alterations were found in five HPRT deficient clones per individual examined for changes in 10 microsatellites, giving an overall mutation rate per allele of 0.3%. Thus, the alterations detected are probably due to background mutations and not to differences in mismatch repair capacity.     

The function of the nuclear matrix attachment region of silkworm rDNA as an autonomously replicating sequence in plasmid and chromosomal replication origin in yeast

Nuclear matrix attachment regions (MARs) play a crucial role in chromatin architecture, gene expression, and DNA replication. Although it is well known that yeast autonomously replicating sequences (ARSs) bind nuclear matrix and MARs also function as ARS elements in yeast, whether a heterologous MAR or ARS element acts as a replication origin in the chromosome has not been elucidated. We previously identified a MAR (rMAR) located in the nontranscribed spacer (NTS) of silkworm Attacus ricini rDNA. We report here that this rMAR contains 10 copies of ARS consensus sequence (ACS) and several DNA unwinding regions. The rMAR employs ARS activity in yeast and a rARS element locates in the 3(') region of the rMAR. Furthermore, we have also revealed that either the rMAR or the rARS element functions as a replication origin in the chromosome. Our results provide the first direct evidence to demonstrate that heterologous rMAR and rARS display chromosomal origin activity, suggesting that the chromosome structure and replication origin of rDNA reserve some common features during evolution.     

Isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae

Summary An 8.6-kb fragment was isolated from an EcoRI digest of Candida albicans ATCC 10261 genomic DNA which conferred the property of autonomous replication in Saccharomyces cervisiae on the otherwise non-replicative plasmid pMK155 (5.6 kb). The DNA responsible for the replicative function was subcloned as a 1.2-kb fragment onto a non-replicative plasmid (pRC3915) containing the C. albicans URA3 and LEU2 genes to form plasmid pRC3920. This plasmid was capable of autonomous replication in both S. cerevisiae and C. albicans and transformed S. cerevisiae AH22 (leu2^–) to Leu⁺ at a frequency of 2.15 × 10³ transformants per pg DNA, and transformed C. albicans SGY-243 (ura3) to Ura⁺ at a frequency of 1.91 × 10³ transformants per g DNA. Sequence analysis of the cloned DNA revealed the presence of two identical regions of eleven base pairs (5TTTTATGTTTT3) which agreed with the consensus of autonomously replicating sequence (ARS) cores functional in S. cerevisiae. In addition there were two 10/11 and numerous 9/11 matches to the core consensus. The two 11/11 matches to the consensus, CaARS1 and CaARS2, were located on opposite strands in a non-coding AT-rich region and were separated by 107 bp. Also present on the C. albicans DNA, 538 by from the ARS cores, was a gene for 5S rRNA which showed sequence homology with several other yeast 5S rRNA genes. A sub-fragment (494 bp) containing the 5S rRNA gene (but not the region containing the ARS cores) hybridized to genomic DNAs from a number of yeast species, including S. cerevisiae, C. tropicalis, C. pseudotropicalis, C. parapsilosis, C. kruseii, C. (Torulopsis) glabrata and Neurospora crassa. The 709-bp ARS element (but not the 5S rRNA gene) was necessary for high-frequency transformation and autonomous plasmid replication in both S. cerevisiae and C. albicans.EMBL/GenBank database accession number: X16634 (5S rRNA)     

A Petunia hybrida chloroplast DNA region, close to one of the inverted repeats, shows sequence homology with the Euglena gracilis chloroplast DNA region that carries the putative replication origin

Summary Three distinct chloroplast (cp) DNA fragments from Petunia hybrida, which promote autonomous replication in yeast, were mapped on the chloroplast genome. Sequence analysis revealed that these fragments (called ARS A, B and C) have a high AT content, numerous short direct and inverted repeats and at least one yeast ARS consensus sequence 5A/TTTTATPuTTTA/T, essential for yeast ARS activity. ARS A and B also showed the presence of (semi-)conserved sequences, present in all Chlamydomanas reinhardii cpDNA regions that promote autonomous replication in yeast (ARS sequences) or in C. reinhardii (ARC sequences). A 431 bp BamHI/EcoRI fragment, close to one of the inverted repeats and adjacent to the ARS B subfragment contains an AT-rich stretch of about 100 nucleotides that show extensive homology with an Euglena gracilis cpDNA fragment which is part of the replication origin region. This conserved region contains direct and inverted repeats, stem-and-loop structures can be folded and it contains an ARS consensus sequence. In the near vicinity a GC-rich block is present. All these features make this cpDNA region the best candidate for being the origin of replication of P. hybrida cpDNA.     

Total gene deletions and mutant frequency of the HPRT gene as indicators of radiation exposure in Chernobyl liquidators

This study was conducted to determine the utility of deletion spectrum and mutant frequency (MF) of the hypoxanthine phosphoribosyl transferase gene (HPRT) as indicators of radiation exposure in Russian Liquidators who served in 1986 or 1987 in the clean up effort following the nuclear power plant accident at Chernobyl. HPRT MF was determined using the cloning assay for 117 Russian Controls and 122 Liquidators whose blood samples were obtained between 1991 and 1998. Only subjects from whom mutants were obtained for deletion analysis are included. Multiplex PCR analysis was performed on cell extracts of 1080 thioguanine resistant clones from Controls and 944 clones from Liquidators. Although the deletion spectra of Liquidators and Controls were similar overall, the Liquidator deletion spectrum was heterogeneous over time. Most notable, the proportion of total gene deletions was higher in 1991–1992 Liquidators than in Russian Controls (χ²=10.5, p=0.001) and in 1993–1994 Liquidators (χ²=8.3, p=0.004), and was marginally elevated relative to 1995–1996 Liquidators (χ²=3.3, p=0.07). This type of mutation has been highly associated with radiation exposure. Total gene deletions were not increased after 1992. Band shift mutations were also increased in the 1991–1992 Liquidators but were associated with increased MF of both Liquidators and Controls (p=0.009), not with increased MF in 1991–1992 Liquidators (p=0.7), and hence are not believed to be associated with radiation exposure. Regression analysis demonstrated that relative to Russian Controls HPRT MF was elevated in Liquidators overall when adjusted for age and smoking status (37%, p=0.0001), and also was elevated in Liquidators sampled in 1991–1992 (72%, p=0.0076), 1993–1994 (22%, p=0.037), and 1995–1996 (62%, p=0.0001). In summary, HPRT MF was found to be the more sensitive and persistent indicator of radiation exposure, but the specificity of total gene deletions led to detection of probable heterogeneity of radiation exposure within the exposed population.     

Validation of the human T-lymphocyte cloning assay — ring test report from the EU concerted action on HPRT mutation (EUCAHM)

The T-cell cloning assay, which enables the enumeration and molecular analysis of 6-thioguanine resistant (HPRT-negative) mutant T-cells, has been extensively used for studying human somatic gene mutation in vivo. However, large inter-laboratory variations in the HPRT mutant frequency (MF) call for further investigation of inter-laboratory differences in the experimental methodology, and development of an optimal but easy uniform cloning protocol. As part of the EU Concerted Action on HPRT Mutation (EUCAHM), we have carried out two Ring tests for the T-cell cloning assay. For each test, duplicate and coded samples from three buffy coats were distributed to five laboratories for determination of MF using six different protocols. The results indicated a good agreement between split samples within each laboratory. However, both the cloning efficiencies (CEs) and MFs measured for the same blood donors showed substantial inter-laboratory variations. Also, different medium compositions used in one and the same laboratory resulted in a remarkable difference in the level of MF. A uniform operating protocol (UOP) was proposed and compared with the traditional protocols in the second Ring test. The UOP (preincubation) increased the CE in laboratories traditionally using preincubation, but decreased the CE in laboratories traditionally using priming. Adjusted for donor, use of different protocols contributed significantly to the overall variation in lnCE (P=0.0004) and lnMF (P=0.03), but there was no significant laboratory effect on the lnCE (P=0.38) or lnMF (P=0.14) produced by the UOP alone. Finally, a simplified version of the UOP using the serum-free medium X-Vivo 10 and PMA was tested in one laboratory, and found to produce a considerable increase in CE. This modified UOP needs to be further evaluated in order to be used for future databases on HPRT MFs in various populations.     

Effects of arsenic exposure on the frequency of HPRT-mutant lymphocytes in a population of copper roasters in Antofagasta, Chile: a pilot study

A pilot biomarker study was conducted to investigate the feasibility of using the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in peripheral blood lymphocytes as a biomarker for detecting genetic effects of arsenic exposure. Blood and urine samples were obtained from workers highly exposed to arsenic in a copper roasting plant in Antofagasta, Chile. Individuals were classified according to their job titles into three potential exposure groups: high, medium, and low. To confirm exposure, arsenic concentration was determined in urine samples. The HPRT mutant frequencies were measured in lymphocytes from 15 individuals ranging in age from 24 to 66 years. The mean mutant frequencies for the three exposure groups were: low (9×10⁻⁶), medium (11×10⁻⁶), and high (24×10⁻⁶). An increased mutant frequency was observed in the highly exposed group, but the response was so slight that it is not likely that this assay will be capable of providing dose–response information across a range of lower, more typical environmental arsenic levels.     

Characterization of a chloroplast DNA sequence from Chlorella ellipsoidea that promotes autonomous replication in yeast

Summary An EcoRI 2.7 kbp fragment from Chlorella ellipsoidea chloroplast DNA (cpDNA) cloned in YIp5 was shown to promote autonomous replication in Saccharomyces cerevisiae. The fragment was localized in the small single copy region close to the inverted repeat. The ARS activity (autonomously replicating sequences in yeast) was found to be confined within a subclone of a ca. 300 bp HindIII fragment. Sequence analysis of this fragment revealed its high AT content and the presence of several direct and inverted repeats and a few elements that were related to the yeast ARS consensus sequence. Electron microscopic studies revealed that this sequence did not coincide with the primary replication origin of chloroplast DNA. The functioning of this sequence as a possible origin of plasmid replication in vivo is discussed. This is the first report on Chlorella cpDNA sequence. re]19850821 rv]19851211 ac]19851216     

Use of recombination techniques to examine the structure of the csg locus of Myxococcus xanthus

Summary The technique of chromosome walking was used to isolate approximately 60 kb of DNA from the region containing the complementation group uncoordinated of Drosophila melanogaster, located in that part of the X chromosome which spans the euchromatin-heterochromatin junction. The cloned DNA can be divided into two distinct regions. The first contains sequences that are low copy number or unique and are largely conserved between strains. The second region is characterized by units repeated in tandem arrays and is polymorphic within, and between, strains. Each repetitive unit is separated by a member of an abundant sequence family, part of which is homologous to the ribosomal type 1 insertion sequence of D. melanogaster. The molecular organization of the cloned DNA was compared with that of sequences isolated from regions of intercalary heterochromatin and also with genes which have been characterized from more conventional euchromatic regions.     

Molecular cloning and nucleotide sequence of the Rhizobium phaseoli recA gene

Summary A recombinant phage carrying the recA gene of Rhizobium phaseoli was isolated from a R. phaseoli genomic library by complementation of the Fec^– phenotype of the recombinant phage in Escherichia coli. When expressed in E. coli, the cloned recA gene was shown to restore resistance to both UV irradiation and the DNA alkylating agent methyl methanesulphonate (MMS). The R. phaseoli recA gene also promoted homologous recombination in E. coli. The cloned recA gene was only weakly inducible in E. coli recA strains by DNA damaging agents. The DNA sequence of the R. phaseoli recA gene was determined and compared with published recA sequences. No LexA-binding site was detected in the R. phaseoli recA upstream region.     

Molecular cloning, sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides

The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA^– mutant of Pseudomonas aeruginosa. Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids. The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium mehloti, Rhizobium phaseoli, and Agrobacterium tumefaciens. An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 by upstream region of the R. sphaeroides recA gene. Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R. sphaeroides is inducible by DNA damage. A recA-defective strain of R. sphaeroides was obtained by replacement of the active recA gene by a gene copy inactived in vitro. The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression. This is the first recA gene from a Gram-negative bacterium that lacks an E. coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.     

Cloning and nucleotide sequence of the Salmonella typhimurium LT2 gnd gene and its homology with the corresponding sequence of Escherichia coli K12

Summary The complete nucleotide sequence of the Salmonella strain LT2 gnd gene for 6-phosphogluconate dehydrogenase was determined. The gene contains 1404 bases and encodes a 468 amino acid polypeptide, which is the same as for Escherichia coli K12. The DNA sequence shows 14.8% difference between the two and the amino acid sequence 3.6% difference. Changes are mostly in the third codon base and most of the amino acid changes are conservative.     

Cloning and nucleotide sequence of the gene braB coding for the sodium-coupled branched-chain amino acid carrier in Pseudomonas aeruginosa PAO

Summary The gene braB, encoding the Na^–-coupled carrier for branched-chain amino acids in Pseudomonas aeruginosa PAO, was cloned on cosmid pMMB34. The cosmid clones carrying the braB gene were identified as those that restored growth at low leucine concentration and Na^–-dependent leucine transport activity to P. aeruginosa PAO3536 defective in the transport of branched-chain amino acids. Determination of the nucleotide sequence of the DNA fragment shows that the braB gene comprises 1311 bp and encodes a hydrophobic protein of 437 amino acids with a calculated M_r of 45279. The hydropathy profile suggests that there exist in the carrier protein 12 hydrophobic segments long enough to traverse the membrane. The amino acid sequence shows a high degree of homology with thebrnQ product, a branched-chain amino acid carrier of Salmonella typhimurium, while no homology in the nucleotide sequences is found in the braB and brnQ genes.     

Common nodABC genes in Nod locus 1 of Azorhizobium caulinodans: Nucleotide sequence and plant-inducible expression

Summary Azorhizobium caulinodans strain ORS571 induces nitrogen-fixing nodules on roots and stem-located root primordia of Sesbania rostrata. Two essential Nod loci have been previously identified in the bacterial genome, one of which (Nod locus 1) shows weak homology with the common nodC gene of Rhizobium mehloti. Here we present the nucleotide sequence of this region and show that it contains three contiguous open reading frames (ORFA, ORFB and ORFC) that are related to the nodABC genes of Rhizobium and Bradyrhizobium species. ORFC is followed by a fourth (ORF4) and probably a fifth (ORF5) open reading frame. ORF4 may be analogous to the nod[ gene of R. leguminosarum, whereas ORF5 could be similar to the rhizobial nodF genes. Coordinated expression of this set of five genes seems likely from the sequence organization. There is no typical nod promoter consensus sequence (nod box) in the region upstream of the first gene (ORFA) and there is no nodD-like gene. LacZ fusions constructed with ORFA, ORFB, ORFC, and ORF4 showed inducible -galactosidase expression in the presence of S. rostrata seedlings as well as around stem-located root primordia. Among a series of phenolic compounds tested, the flavanone naringenin was the most efficient inducer of the expression of this ORS571 nod gene cluster.     

The Drosophila Tumorous lethal hematopoietic oncogene is a dominant mutation in the hopscotch locus

The Drosophila Tumorous-lethal (Tum-l) mutation acts as an activated oncogene, causing hematopoietic neoplasms, overproliferation, and premature differentiation. Tum-l is a dominant mutation in the hopscotch (hop) locus, which is required for cell division and for proper embryonic segmentation. The Tum-l temperature-sensitive period for melanotic tumor formation includes most of larval and pupal development.     

Amino acid sequence and function of rebredoxin from Desulfovibrio vulgaris Miyazaki

Rubredoxin was purified from Desulfovibrio vulgaris Miyazaki. It was sequenced and some of its properties determined. Rubredoxin is composed of 52 amino acids. It is highly homologous to that from D. vulgaris Hildenborough. Its N-methionyl residue is partially formalated. The millimolar absorption coefficients of the rubredoxin at 489 nm and 280 are 8.1 and 18.5, respectively, and the standard redox potential is +5 mB, which is slightly higher than those of other rubredoxins. Rubredoxin, as well as cytochrome c-553, was reduced with lactate by the action of lactate dehydrogenase of this organism, and the rection was stimulated with 2-methyl-1, 4-naphthoquinone. It is suggested that rubredoxin, in collaboration with membraous quinone, functions as natural electron carrier for cytoplasmic lactate dehydrogenase of this organism, whereas cytochrome c-553 plays the same role for periplasmic lactate dehydrogenase.